A schematic of the clotting cascades is shown in FIG. 5(A). In the figure the various clotting factors are indicated by their Roman numeral (i.e., factor VII is indicated by VII). The intrinsic pathway (also referred to as the contact pathway of blood coagulation) is initiated when contact is made between blood and certain artificial surfaces. The extrinsic pathway (also referred to as the tissue factor pathway of blood coagulation) is initiated upon vascular injury which leads to exposure of tissue factor (TF) (also identified as factor III). The dotted arrow represents a point of cross-over between the extrinsic and intrinsic pathways. The two pathways converge at the activation of factor X to Xa. Factor Xa has a role in the further activation of factor VII to Vila. Active factor Xa hydrolyzes and activates prothrombin to thrombin. Thrombin can then activate factors XI, VIII and V furthering the cascade. Ultimately, the role of thrombin is to convert fibrinogen to fibrin, which forms clots.
Fibrinogen is the most abundant coagulation protein in blood. The formation of a fibrin clot from fibrinogen is the terminal step in the coagulation cascade. Soluble fibrin monomers, which are created when thrombin cleaves fibrinogen, spontaneously polymerize to form a three dimensional network of insoluble fibrin fibrils. Clotting of fibrinogen by thrombin is one of the few steps in the clotting cascade that does not require calcium ions. The resulting fibrin clot structure can be further stabilized via covalent cross-linking of the fibrils through the action of the transglutaminase enzyme, factor XIIIa (FIG. 5 (B)) [18].
Hemorrhage is a major complication of both naturally occurring factor deficiencies such as hemophilia, and anticoagulant therapy. Hemorrhagic episodes can result in significant patient morbidity and in rare cases, mortality. Even in patients with well-controlled stable anticoagulant therapy, emergent circumstances may arise that necessitate immediate reversal of anticoagulant status.
Rapid normalization of abnormal coagulation has generally relied on either replacement of missing factors or administration of specific antidotes [1]. A major limitation of this approach is that transfusion with human-derived products has the potential for transmission of infectious disease. Furthermore, most anticoagulant drugs, including most newly approved anticoagulant drugs, as well as some in development, lack effective antidotes [2]. In vitro studies of the effects of recombinant factor Vila (rFVIIa) [5-7] and off-label use in vivo [8-11] have indicated that rFVIIa might have a role as a general method of reversing anticoagulant therapy. Use of rFVIIa may be associated with thromboembolic adverse events [12,13]. Currently, the primary factors limiting use of rFVIIa as a universal procoagulant agent are the potential liability associated with off-label use, and the extreme expense associated with this drug.
Heparin is a naturally occurring sulfated polysaccharide. It functions as an anticoagulant by indirectly inhibiting the enzymatic activity of factor Xa and thrombin through its ability to enhance the action of the plasma anticoagulant protein, antithrombin. Heparin is widely used as a clinical anticoagulant for such indications as cardiopulmonary bypass surgery, deep vein thrombosis, pulmonary thromboembolism, arterial thrombosis, and prophylaxis against thrombosis following surgery. Therapeutic plasma concentrations of heparin are generally 0.2-0.7 units/ml. The effects of heparin can be rapidly reversed using the specific antidote protamine [1].
Low molecular weight heparins (low MW heparins), such as enoxaparin, are widely used anticoagulant drugs. Low MW heparins are size-fractionated to obtain preparations in which the heparin polymers are shorter and less heterodisperse than unfractionated heparin. Low MW heparins act primarily as factor Xa inhibitors, as they enhance antithrombin's anticoagulant effect toward factor Xa to a much greater extent than toward thrombin. Low MW heparins are widely used for longer-term anticoagulant therapy to prevent deep vein thrombosis and have certain advantages over unfractionated heparin. Therapeutic plasma concentrations of low MW heparins are generally 0.2-2 units/ml. Low MW heparins have plasma half-lives of 4-13 hours, resulting in prolonged anticoagulation even if the drug is discontinued when bleeding occurs. There is no generally accepted antidote available to reverse anticoagulation with low MW heparin.
COUMADIN® (warfarin sodium) is an oral anticoagulant drug that reduces the effective concentration of several coagulant proteins in plasma. It is widely used as long-term therapy for prevention of arterial and venous thrombosis. The most important adverse effect of COUMADIN® therapy is hemorrhage, particularly into the central nervous system. COUMADIN® therapy is typically monitored by a plasma clotting test whose readout is the International Normalized Ratio (INR). For patients receiving COUMADIN®, anticoagulant status can be immediately reversed with plasma transfusion should serious bleeding occur, but this therapy is expensive and carries risks of transfusion reactions and transmission of infectious diseases. The anticoagulant effect of COUMADIN® can be more slowly reversed with Vitamin K therapy [1].
Argatroban is an oral anticoagulant drug that is a small molecule inhibitor of thrombin. It is approved for anticoagulant therapy in patients at risk for thrombosis who cannot be treated with heparin. Therapeutic plasma concentrations are about 1 μg/ml. There is no available antidote to reverse anticoagulation with argatroban.
Rivaroxaban is an experimental anticoagulant drug under development. It functions as a small molecule inhibitor of factor Xa. It is used for prevention of thrombotic complications of orthopedic surgery. It has a plasma half-life of 7-10 hours [21]. There is no available antidote to reverse anticoagulation with rivaroxaban.
Hemophilia A is an inherited or acquired deficiency of coagulation factor VIII (FVIII) and is associated with risk of severe bleeding. Patients with hemophilia A who develop a serious bleeding episode can be treated with purified human FVIII, but this therapy is quite expensive, costing thousands of dollars per episode [3]. About one-third of patients who receive repeated doses of human FVIII will develop inhibitory antibodies to the drug, which may prevent its further use in that patient. Bleeding episodes in hemophilia patients with such inhibitory antibodies may be treated with high doses of recombinant human factor Vila (rFVIIa); treating one bleeding episode with this drug can cost in excess of $70,000 [3,4].
Hemophilia B is an inherited or acquired deficiency of coagulation factor IX (FIX) and is associated with risk of severe bleeding. Clinical presentation and treatment are similar to that for Hemophilia A, except that injection of purified FIX is used to treat bleeding in these patients [3].
The Prothrombin Time (PT) test is widely used to monitor oral anticoagulation therapy by COUMADIN®, as a general screening test for the blood clotting system, and as the basis for specific Factor assays. Clotting times obtained with the PT are primarily dependent on the plasma levels of the vitamin K-dependent coagulation Factors II (prothrombin), VII, and X, and on the levels of two non-vitamin K-dependent proteins, Factor V and fibrinogen. The PT test is accomplished by mixing citrated plasma samples with a thromboplastin reagent and measuring the time to clot formation. The ISI value of a thromboplastin reagent is used to calculate the International Normalized Ratio (INR) for patient plasma samples; the more sensitive a thromboplastin reagent is to the changes induced by oral anticoagulant therapy, the lower its ISI value. The INR is calculated by first dividing the patient's PT value by the mean PT value for 20 or more normal plasmas. This PT ratio is then raised to the ISI power, yielding the INR value, which in turn, is used by the treating physician to adjust the drug dose. The introduction of the INR reporting system has vastly improved the standardization of monitoring of oral anticoagulant therapy, and can be credited with decreasing bleeding complications for oral anticoagulant therapy [20]. Normal plasma is defined as an INR of about 1.0. Therapeutic INR values are generally in the range of 2.0-3.5.
Polyphosphate (polyP) is a negatively charged, linear polymer of phosphate units linked by high energy phosphoanhydride bonds [14]. Dense granules of human platelets contain millimolar levels of polyP (with chain lengths of approximately 75 phosphate units) [15]. PolyP is released from platelets in response to stimulation by thrombin [17] and is cleared from plasma presumably due to degradation by plasma phosphatases [17]. We recently reported that polyphosphate is a potent hemostatic regulator, accelerating blood coagulation by activating the contact pathway of blood clotting, promoting the activation of factor V, and abrogating the function of tissue factor pathway inhibitor (TFPI) [17]. These combined effects result in a shift in the timing of thrombin generation without changing the total amount of thrombin generated. Polyphosphate also delays fibrinolysis through a thrombin-activatable fibrinolysis inhibitor (TAFI)-dependent mechanism, presumably as a consequence of an earlier burst in thrombin generation [17].
Polyphosphate, radiolabeled with 99mTc, administered by injection, has been used as a radiopharmaceutical for skeletal imaging [16].